| Schizothorax prenanti belongs to Cypriniformes, Cyprinidae, the abdomen fishsubfamily (Schizothoracinae), Schizothorax is (Schizothorax). Located in the upperreaches of the Yangtze River, Ren River, Dadu River, Minjiang River, Qingyi, theJinsha River, Emirates Water and the Lower Reaches of the Wujiang River and otherwaters. The Qi port Schizothorax the bottom-dwelling fish, living in the lower watertemperature environment, living in Jihuan flow at the junction, a short distance fromreproductive migratory phenomenon. High oxygen content, the mountains and riversare cold water fish.Trypsin digestion enzymes that belong to the serine protease family,, is a majoralkaline proteolytic enzymes in the animal digestive tract. TPretrypsinogen equal to247amino acids, to remove the signal peptide of15amino acids (Aa) formtrypsinogen, trypsinogen N-terminal hydrolysis down trypsinogen the activationpeptide. after the formation of trypsin.In this study, from the molecular level to clarify Schizothorax prenanti trypsinmechanism to establish certain basic molecular biology. Schizothorax prenanti farmingprocess of bio-feed and fishery waste utilization theory or basis in reality, while thestudy is to elucidate the molecular evolution of the vertebrate trypsinogen precursorgene academic.In this study, the RACE technique to clone Schizothorax prenanti trypsinogenprecursor protein gene, and its nucleotide sequence and deduced amino acid sequenceand predicted protein structure analysis.Nested middle fragment primer design in the multiple than trypsinogen precursorgene of cyprinids from Schizothorax prenanti pancreas tissue mRNA for thepull-mode amplified Schizothorax prenanti trypsin the middle of the original precursorfragment sequences. The middle fragment cDNA sequences to design two specific3'RACE primers, two the5'RACE specific primers, resulting in the sequence of the3'and5' sequence of the original precursor of the mullet trypsin. After stitchingSchizothorax prenanti trypsinogen precursor full-length cDNA. Verified according tothe spliced cDNA was designed primers, proven results. Schizothorax prenanti trypsinogen precursor cDNA from full-sequence information for: cDNA full length931bp, of which the5'untranslated region of63bp, the protein coding region of741bp3' UTR127bp.Schizothorax prenanti trypsinogen precursor protein gene open reading frameencoding246amino acids, including the signal peptide of15amino acids and eightamino acid activation peptide. The sequence has a conserved sequence of serineproteases: The sequence has the conserved sequence of serine proteases:(1) containsthe conserved His (H63), Asp (D107) and Ser (S200) active triad (2) has the typicalconservative substrate binding site Asp (D194), the residues located in the bottommaterial combined with the bottom of the ditch, and by the charge effect and stablesubstrate cleavage site, Lys or Arg residues, and thus determine the specificity oftrypsin (3) open reading frame encoding256amino acids, including a15amino acidsignal peptide and eight amino acid activation peptide.(4) has six disulfide bonds, thebasic structure of the gene and cloned from other vertebrate trypsinogen precursor ofgene structure.Schizothorax prenanti trypsinogen precursor amino acid sequence has strongsimilarity with the many animals trypsinogen precursor gene. In particular, the role ofsubstrate and the active triad is completely conserved. In addition to the substrate sitesof action in the vicinity of amino acid residues with conservative, as well as serinefamilial form three disulfide bonds of the six cysteine residues in all eukaryotic animalare conserved in vertebrates generally have six disulfide bonds, amino acid residues ofthese sites are also very conservative. Similarities and trends in the opposite sexcompared with DNAstar software MgeAlign on several vertebrate trypsin. In additionto those particular sites, the composition of the vertebrate trypsinogen precursor aminoacid is not particularly conservative, Schizothorax prenanti on the trypsinogenprecursor amino acid sequence with China Spinibarbus Spinibarbus sinensis,trypsinogen precursor similar rates of up to90%, followed by86%of the zebrafishDanio similar rate, with mullet Myxocyprinus asiaticus,85%.Phylogenetic analysis farthest trypsinogen precursor the overall performance ofthe phylogenetic relationships among the different purposes, belong to thephylogenetic relationship within species, the same mesh internal medicine, followed bythe phylogenetic relationships among The evolutionary trend is reflected in the trypsin:followed by differentiation of the cartilaginous fish, bony fish, amphibians and birds,the final differentiation of mammals. Trypsinogen precursor gene predicted amino acid sequence analysis, the basicparameters of the protein, its atoms C1146H1792N320O36S18, molecular weight Mw=26469.8Da, isoelectric point pl=6.85. Amino acid composition, the highest content ofamino acids serine (Ser)(13%); followed by glycine (Gly)(8.9%), asparagine (Asn)and (Ala)7.3%; the lowest levels of amino acids is histidine acid (His) and tryptophan(Trp) only l.6%. On the whole, the basic amino acids (His,Lys, Arg)(8.6%), acidicamino acids (Asp, Glu) accounted for7%, with pancreatic The proteasome isconsistent with a basic amino acid.Schizothorax prenanti trypsinogen precursor protein secondary structure ismainly composed by the four forms, namely a-helix (Alpha helix) accounted for10.16%; random coil (random coil) accounted for60.16%: extended strand (extendedstrand) accounting for29.67%. It can be seen that the crack in Schizothorax prenantion the trypsinogen precursor protein secondary structure extension chain and randomcoil proportion is quite high, extended strand are mainly located between the a-helixand random coil.Schizothorax prenanti trypsinogen precursor expression in pancreas and foregut,midgut and hindgut highest amount, followed by expression in the liver, kidney, spleen,heart, brain expression less in the gills in the expression of basic no. |
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